During the last grant period, we made a novel observation: Treatment of melanoma patients with a vaccine consisting of autologous tumor cells conjugated to the hapten dinitrophenol (DNP) results in the development of inflammatory responses in metastases. Histologically these responses are characterized by infiltration of the tumors with T Iymphocytes. In several of these patients, we observed clinically-defined tumor regression, including a complete remission of extensive lung metastases. Furthermore, a high percentage of patients who received the vaccine after resection of bulky lymph node metastases have remained alive and disease-free. In this renewal application, we propose to extend the clinical trials and to conduct laboratory studies of the T cells infiltrating DNP-vaccine-treated metastases. Patients with measurable metastases will be treated by a new, more intensive program that involves weekly administration of vaccine preceded by a single dose of cyclophosphamide. Metastases that developed inflammation will be excised and cryopreserved. We will determine whether the infiltrating T cells have a characteristic phenotype or lymphokine profile; the latter will be studied by a polymerase chain reaction (PCR)- based technique. We hypothesize that T cells found in regressing tumors will have the TH1 profile, producing lL-2 and gamma interferon, but not the anti-inflammatory lymphokines, lL-4 and lL-10. PCR will also be used to characterize the T cell receptor variable (TCR-V) regions on these T cells; we predict that the TCR-V repertoire will be restricted, suggesting that the T cells are responding to a limited number of melanoma antigens. In another project, those T cells will be cloned by limiting dilution, and the clones will be studied for autologous melanoma reactivity, both cytotoxicity and lymphokine production. Finally, we will study clones of PBL and T cells from DNP-vaccine injection sites to determine whether DNP- modified melanoma cells elicit responses that are distinct from those elicited by DNP-modified lymphocytes. We expect these studies to lead to advances in the treatment of human melanoma and a better understanding of the immunobiology of that disease.